Lecture 17

Question 1

what type of genetic inheritance is most likely occurring if we have affected phenotypes in the first and third generation?

Answer 1

autosomal recessive disorder

Question 2

what did they find when they performed whole exome sequencing on the grandfather and the younger boy?

Answer 2

the variant is a C → T (homozygous in both the grandfather and the nephews and heterozygous in the parents) localized in the POPDC1 gene, which is not a causative gene because it is not associated to any human disorder

Question 3

what family of genes does POPDC1 belong to?

Answer 3

Popeye domain

Question 4

what is the Popeye domain of genes?

Answer 4

a motif that is highly conserved among species and has a high affinity for cAMP → has a site for the binding of cAMP and for the binding to a voltage-gated channel for K+ called TREK1

Question 5

what can the function of POPDC1 modulate?

Answer 5

the function of TREK1 protein

Question 6

what does the Popeye domain encode for?

Answer 6

a family of transmembrane proteins expressed in skeletal and cardiac muscle

Question 7

what was the first experiment the scientist did to understand the physiological function of the protein?

Answer 7

performed a muscular biopsy of the grandfather and young boy and analyzed the tissue through immunostaining

Question 8

what did they first find about the difference in POPDC1 & 2 presence in healthy individuals and the two patients?

Answer 8

POPDC1 & 2 are present on the membranes of healthy individuals, but we have a reduction of theres proteins on the membranes of the patients → they have an alteration of the localization of the protein

Question 9

next they analyzed the expression level through Western blots - what did they discover?

Answer 9

there was no difference in the expression levels of the patents and healthy individuals, as well as in the expression of other proteins such as caveolin and dystrophin → abnormal localization is linked to the accumulation of the protein in the perinuclear zone

Question 10

the Popeye domain has a high affinity for what?

Answer 10

cAMP

Question 11

what did scientist find in the WT sequence in these two patients?

Answer 11

we have a Serine substituted with a Phenylalanine → this amivoacidic mutation can affect the affinity for cAMP

Question 12

what was discovered about the interaction of the POPDC1 & 2 domains in regards to their interaction with TREK1?

Answer 12

there were not any changes observed so this mutation does not affect this interaction

Question 13

what is the TREK1 domain, and how can we measure it?

Answer 13

a voltage gated channel - we are able to perform electrophysiological studies exploiting patch clamp experiments which are useful to investigate if there are changes in the electrical activity of the channel

Question 14

what was used to study the properties of the voltage gated channel?

Answer 14

xenopus oocytes

Question 15

what did the xenopus oocytes show us about TREK1 and the voltage gated channels?

Answer 15

1. POPDC1 increases the activity of TREK1, so we have the hyperactivity of TREK1 protein if there is a mutation in POPDC1
2. we have a higher current in situations where we have mutated POPDC1 probably due to a gain of function of the protein

Question 16

after the studies in xenopus oocytes, what did scientists study next?

Answer 16

HL-1 cells

Question 17

what are HL-1 cells?

Answer 17

an immortalized cell line of human cardiomyocytes

Question 18

when HL-1 cells were compared to POPDC1 WT and mutated cells, what did they discover when patch clamp analysis was performed to study the electrophysiological activity of the cells?

Answer 18

alterations in the hyper polarization and in the action potential duration → with the reduction of the duration in the action potential in the presence of mutated POPDC1

Question 19

how did the presence of the mutated POPDC1 alter the action potential duration?

Answer 19

there was not a direct mutation of the voltage gated channel, but there was an indirect effect due to an altered function of the POPDC1 protein that can modulate the TREK1 leading to a GOF of the potassium voltage gated channel

Question 20

how does an alteration of the cardiac action potential occur?

Answer 20

a higher hyperpolarization of the cell can lead to an increase of the sodium channel (GOF), leading to an alteration of the cardiac action potential and therefore an atrial-ventricular block

Question 21

why are zebrafish a good model in order to study cardiac function in this case?

Answer 21

we can have a visual characterization of the cardiac alteration and POPDC1 is expressed in both cardiac and skeletal muscle

Question 22

besides altering the alteration of the cardiac action potential, what else can POPDC1 effect?

Answer 22

can also effect the development of the skeletal muscle by altering the orientation and the organization of skeletal fibers

Question 23

what did they see with the cardiac phenotype of the zebrafish with a POPDC1 mutation?

Answer 23

a reduction of heart rate (fish are bradycardic), leading to sudden arrest and the block that is present in the human patients

Question 24

even though we have a single gene that is segregated with an altered phenotype, it was not sufficient to demonstrate that the gene was causative - why?

Answer 24

due to the fact that we don’t know anything about that gene - it was never associated to another human disorder, and it is essential first to characterize the clinical phenotype

Question 25

throughout the familial study as well as the research done with zebrafish and oocytes, what four things did they discover?

Answer 25

1. mutation on POPDC1 gene can cause muscular and cardiac disorders
2. zebrafish knock-in model shows either muscular or cardiac phenotype
3. POPDC1 has a biological role in the modulation of physiological activity of TREK1
4. POPDC1 and TREK1 are regulated by cAMP